This project is focused on understanding of why HIV-positive versus HIV-negative individuals are more likely to progress to human papillomavirus 16 (HPV 16)-associated anal high-grade squamous intraepithelial lesions (HSIL) and anal cancer. Although HPV-associated anal HSIL and cancers from HIV-positive and HPV- negative individuals are histopathologically similar, epithelial exposure to HIV, before or after infection with HPV, may lead to cellular changes in the epithelium that are more likely to result in cellular transformation, thus increasing the incidence of anal cancer in this population. Several studies have suggested that exogenous HIV proteins (e.g. tat and nef) interfere with cellular proteins and pathways that are known to be dys-regulated in HPV 16-associated cancers. One way that tat and nef interfere with cellular proteins and their corresponding pathways is through modulation of microRNA (miRNA) expression. The human genome encodes over 3,707 miRNAs, which frequently target many genes related to cancer development or prevention. Differentially-expressed miRNAs have been detected in several types of tumors and at different stages of those tumors. We hypothesize that HIV proteins modulate the miRNA profile of anal epithelial cells leading to dys-regulation of cellular pathways that regulate host oncogenes and tumor suppressor genes. Co-infection with HPV 16 further enhances this dys-regulation of cellular miRNAs and their corresponding pathways. Partnered with the Anal Neoplasia Clinic, Research and Education (ANCRE) and the Sandler Functional Genomics Facility at UCSF, we will perform miRNA profiling in archived anal biopsies from HIV- positive and HIV-negative individuals. We will compare the cellular miRNA profiles in tissues of different grades of pathology, including normal tissues, HSIL and cancer using the HTG EdgeSeq miRNA whole transcriptome assay, a novel technique that measures the expression of 2,275 human miRNAs. This will allow us to delineate the differences and similarities in HPV 16-positive anal tumors that arise from HIV-positive versus HIV-negative individuals. In addition we will use an anal epithelial in-vitro model to: 1) Identify HIV proteins that lead to dys-regulation of cellular miRNAs, 2) Validate the expression of a subset of miRNAs of interest in the setting of HIV/HPV 16 infection alone and during co-infection, and 3) Demonstrate the effects of individual miRNAs on cellular pathways and proteins that are involved in HPV 16-associated anal cancer progression. This will be the first study to establish a miRNAome specific to anal cancer in the setting of HIV infection. The identification of HIV-associated cellular miRNAs that are involved in HPV 16-associated anal cancer progression could lead to development of new diagnostic/prognostic markers, as well as therapies to treat anal cancers, particularly in HIV-positive individuals.